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Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function.

ABSTRACTProteostasis of the endoplasmic reticulum (ER) is maintained by coordinated action of two major catabolic pathways: proteasome-dependent ER-associated degradation (ERAD) and less characterized lysosomal pathways. Recent studies on ER-specific autophagy (termed “reticulophagy”) have highlighted the importance of lysosomes for ER proteostasis. Key to this process are proteins termed reticulophagy receptors that connect ER fragments and Atg8-family proteins, facilitating the lysosomal degradation of both native and aberrant ER proteins in a relatively nonselective manner. In contrast, our recent work identified TOLLIP as a novel type of cargo receptor specifically dedicated to the lysosomal degradation of aberrant ER membrane proteins. The clients of TOLLIP include an engineered model substrate, which mimics an ER-retained aberrant membrane protein, and motor neuron disease-linked misfolded mutants of VAPB and BSCL2/Seipin. TOLLIP acts as a receptor to connect these aberrant ER membrane proteins and phosphatidylinositol-3-phosphate (PtdIns3P) by recognizing the former through its misfolding-sensing intrinsically disordered region (IDR) and ubiquitin-binding CUE domain, and the latter through its C2 domain. These interactions enable PtdIns3P-dependent vesicular trafficking of aberrant membrane proteins to lysosomes without promoting reticulophagic turnover of bulk ER.

AbstractA novel method, called Advanced Multi-Step Brazing, was developed to fabricate a new type of divertor heat removal component with W armor and an oxide-dispersion-strengthened copper (GlidCop®) heat sink in the initial phase of our work. Later, a new type of divertor heat removal component, which has a rectangular-shaped cooling channel with a V-shaped staggered-rib structure in the GlidCop heat sink, was developed. This new component showed an extremely high heat removal capability during a ~30 MW/m2steady-state heat loading condition in our previous work. In this work, the new component was installed in the divertor strike position of the Large Helical Device and exposed to neutral beam injection–heated plasma discharges with 1180 shots (~8000 s) in total. Though submillimeter-scale damage, such as unipolar arc trails and microscale cracks, was identified on the W surface, the extremely high heat removal capability did not show any sign of degradation over the experimental period. On the other hand, remarkable sputtering erosion and redeposition phenomena, due to the strong influx of the divertor plasma, was confirmed on the W armor.

A series of six dications composed of pure hydrocarbons with one to six non-substituted 9,10-anthrylene units end-capped with two dibenzotropyliums were designed and synthesized to elucidate the electronic properties of huge oligo(9,10-anthrylene) backbones. Their structures were successfully determined by X-ray analyses even in the case of eight planar 14π-electron units, revealing that all dications adopt almost orthogonally twisted structures between neighboring units. Spectroscopic and voltammetric analyses show that neither the significant overlap of orbitals nor the delocalization of electrons between 14π-electron units occurs due to the orthogonally twisted geometry even in solution. As a result, sequential oxidation processes were observed with the reversible formation of multivalent cations with the release of the same number of electrons as the number of anthrylene units. Upon two-electron reduction, a closed-shell butterfly-shaped form was obtained from the dication containing one anthrylene unit, whereas open-shell twisted biradicals were isolated as stable entities in the cases of derivatives containing three to six anthrylene units. Notably, from the derivative with two anthrylene units, a metastable open-shell isomer was obtained quantitatively and underwent slow thermal conversion to the most stable closed-shell isomer (Ea= 23.1 kcal mol–1). There is a drastic change in oxidation potentials between two neutral species (ΔE= 1.32 V in CH2Cl2). Since the present dications were regenerated upon oxidation of the isolated reduction products, these systems may contribute to the development of advanced response systems capable of switching color, magnetic properties, and oxidative properties by using a “cation-capped orthogonal geometry”.

This paper proposes a new approach to estimate the inertia of the 60 Hz Japanese power system from the background signals of phasor measurement units. Regarding the constraint boundaries, the entire system is divided into two control areas, which can be represented by an equivalent two-generator model based on small-signal stability analysis. The ambient inertia of each area, including both generation and demand sides can be estimated by measuring the corresponding phase difference and interchange power with respect to the extracted proportions of the frequency response, inter-area oscillation, and synchronizing power. Then, the sum of the estimated inertia for each area gives the total inertia of the entire 60 Hz system. The method was validated on the benchmark model of the Japan transmission network, and its sensitivity is evaluated by conducting various system operations on generator control effects. The efficiency and robustness were verified through real measurements under diverse system conditions. The results confirm that the developed approach can accurately estimate the system inertia during normal operation, enabling nearly continuous inertia tracking trajectory.

'LISTEN TO MY WORDS.' It's a phrase parents use all the time. But what are we really saying? When it comes to raising confident girls, we may be giving them [...]

From the viewpoint of the development of molecular response systems, stimulus-induced switching of multi-conformational/multi-configurational overcrowded ethylenes are interesting, whose properties could be manipulated by understanding the detailed isomerization paths. Anthraquinodimethane (AQD) ring-flip is usually a very fast process, and thus less studied experimentally. Herein, we studied AQDs with dibenzo- and tribenzocycloheptatrienylidene units, which have large enough steric hindrance to retard the AQD ring-flip to allow determination of the ΔG≠value experimentally. Their thermal isomerization was also scrutinized using the artificial force induced reaction method to elucidate the intermediates. Based on the structural unsymmetry in a newly prepared AQD, one of the isomers that undergoes a reversible conformational change via AQD ring-flip was isolated and analyzed by X-ray for the first time.Understanding the mechanism of conformational/configurational isomerism of sterically congested alkenes is of great importance to develop novel molecular response systems. Herein, through the careful study on overcrowded anthraquinodimethane (AQD) derivatives including unsymmetrical compounds, we found AQD ring-flip is the key process to make stimuli-responsive materials with multi-conformational/multi-configurational isomers enabling the switching of their structures and physical properties.

Tritium is a low energy beta emitter and is discharged into the aquatic environment primarily in the form of tritiated water (HTO) from nuclear power plants or from nuclear fuel reprocessing plants. Although the biological effects of HTO exposures at significant doses or dose rates have been extensively studied, there are few reports concerning the biological effects of HTO exposures at very low dose rates. In the present study using a hyper-sensitive assay system, we investigated the dose rate effect of HTO on the induction of mutations. Confluent cell populations were exposed to HTO for a total dose of 0.2 Gy at dose rates between 4.9 mGy/day and 192 mGy/day by incubating cells in medium containing HTO. HTO-induced mutant frequencies and mutation spectra were then investigated. A significant inflection point for both the mutant frequency and mutation spectra was found between 11 mGy/day and 21.6 mGy/day. Mutation spectra analysis revealed that a mechanistic change in the nature of the mutation events occurred around 11 mGy/day. The present observations and published experimental results from oral administrations of HTO to mice suggest that a threshold dose-rate for HTO exposures might exist between 11 mGy/day and 21.6 mGy/day where the nature of the mutation events induced by HTO becomes similar to those seen in spontaneous events.

An intramolecular [4+2] cycloaddition of 1-(9-anthrylchlorophosphino)-1,4-diphenylbut-1-en-3-yne produced the 1-(chlorophosphino)-1,4-diphenyl-1,3-butadiene derivative incorporated with a dibenzobarrelene skeleton, the following reactions of which gave the corresponding secondary phosphine oxide and phenylphosphine sulfide. The secondary phosphine oxide was deprotonated with butyllithium followed by treatment with methyl iodide to give methylphosphine oxide. The phenylphosphine sulfide was desulfurized with P(NMe2)3to give the corresponding phenylphosphine, which was converted to the oxide and selenide and AuCl and BH3complexes. Their structures were determined by X-ray crystallography. These compounds show blue fluorescence in solution (FF= 0.49–0.86) and the solid state (FF= 0.02–0.72) in a narrow range (?em= 442–463 nm) except the phenylphosphine selenide with a low fluorescence efficiency. Their photophysical properties were considered theoretically. The secondary phosphine oxide and the methylphosphine oxide were optically resolved by means of HPLC equipped with a chiral column, and the chiroptical properties were investigated to show weak circular polarized luminescence (CPL) of |gem| = 2.8–8.8 × 10-4.Intramolecular [4+2] cycloaddition of 1-(9-anthrylchlorophosphino)-1,4-diphenylbut-1-en-3-yne produced the 1-(chlorophosphino)-1,4-diphenyl-1,3-butadiene incorporated with a dibenzobarrelene, from which the corresponding secondary phosphine oxide, methylphosphine oxide, phenylphosphine and the chalcogenides, and AuCl and BH3complexes were synthesized. They show blue fluorescence in solution and the solid state. The optically resolved secondary phosphine oxide and the methylphosphine oxide showed CPL of |gem| = 2.8–8.8 × 10-4.

A triarylamine bearing three benzofuran rings was designed and successfully prepared in 64% yield. It exhibited absorption at 385 nm, and blue emission at around 440 nm with a fluorescence quantum yield of 0.57. This compound showed high heat resistance and reversible one-electron oxidation. Upon one-electron chemical oxidation, a near-infrared absorption was revealed due to a persistent radical cation species. DFT calculations also supported the experimental data.A triarylamine bearing three benzofuran rings was designed and successfully prepared in 64% yield. It exhibited absorption at 385 nm, and blue emission at around 440 nm with a fluorescence quantum yield of 0.57. This compound showed high heat resistance and reversible one-electron oxidation. Upon one-electron chemical oxidation, a near-infrared absorption was revealed due to a persistent radical cation species. DFT calculations also supported the experimental data.

A two-pot synthesis of thieno[3,2-b]indole from 2,5-dibromothiophene is described. A halogen dance of 2,5-dibromothiophene was performed with LDA, and subsequent Negishi coupling was performed with 2-iodoaniline derivatives to provide the corresponding coupling products. The resulting two bromo groups have different reactivities, which were utilized for the one-pot Suzuki–Miyaura coupling/intramolecular Buchwald–Hartwig amination to produce thieno[3,2-b]indole via an assisted tandem catalysis that involved in situ ligand exchange.

We go to the gym, eat our greens and aim for eight hours of sleep at night. But break out the bubbly, and we slip from sipping to chugging. Is [...]

Get fitter than ever--and have a rock-hard body to match. Join the triathlon revolution! Move over, marathons. There's a fun new way for more people to get maximum benefits from [...]

Climbing flights is the latest A-lister workout ! And no wonder--it torches calories in no time, while toning your legs and butt STAIRWAY TO HEAVEN? After 12 storeys up a [...]

Why cupcakes? After spending 15 years teaching high school math, I reflected on what I really wanted to do. I've always baked for relaxation, so I directed my entrepreneurial spirit [...]

UFC's street-style fights are winning fans in and outside of the ring. Here's why more and more women are loving mixed-martial-arts workouts The martial arts student spars with her opponent. [...]

Could barely-there footware fix your posture, back and joint problems? We try it on for size I'M A SUCKER FOR SHOES. There's a shoe for everything, I believe. Even if [...]

Highly strained hydrocarbons with two di/tribenzocycloheptatriene units were designed as electrochromic overcrowded ethylenes that undergo reversible interconversion with stable dicationic dyes. Due to severe steric repulsion, two configurational isomers (anti,anti-folded and syn,anti-folded forms) were isolated as stable entities. Photo- and thermal interconversion of these isomers proceeded cleanly: one-way photoisomerization occurred from anti,anti- to syn,anti-form and one-way thermal isomerization was observed from syn,anti- to anti,anti-form. Even though both isomers undergo two-electron oxidation into the same twisted dications, quite different oxidation potentials enable completely selective oxidation of syn,anti-isomers. Thus, the present multiconfigurational strained hydrocarbons are capable of switching of activation/deactivation of their electrochromic properties by light/heat.

The onset of carious lesions and periodontal disease originates in plaque formed from dental bacteria. Appropriate plaque control is therefore important. Dental floss is one instrument used to control plaque. The influence of guardians is an important factor in whether children use dental floss regularly. However, the use of dental floss among school-age children and the relationship between use and family background has yet to be examined.

A solid solution of GaN and ZnO (GaN:ZnO) is promising as a photocatalyst for visible-light-driven overall water splitting to produce H2. However, several obstacles still exist in the conventional preparation procedure of GaN:ZnO. For example, the atomic distributions of Zn and Ga are nonuniform in GaN:ZnO when a mixture of the metal oxides, i.e. Ga2O3and ZnO, is used as a precursor. In addition, GaN:ZnO is generally prepared under a harmful NH3flow for long durations at high temperatures. Here, a facile synthesis of GaN:ZnO with homogeneous atomic composition via a simple and safe procedure is reported. A layered double hydroxide (LDH) containing Zn2+and Ga3+was used to increase the uniformity of the atomic distributions of Zn and Ga in GaN:ZnO. We employed urea as a nitriding agent instead of gaseous NH3to increase the safety of the reaction. Through the optimization of reaction conditions such as heat treatment temperature and content of urea, single-phase GaN:ZnO was successfully obtained. In addition, the nitridation mechanism using urea was investigated in detail. NH3released from the thermal decomposition of urea did not directly nitride the LDH precursor. X-ray absorption and infrared  spectroscopies revealed that Zn(CN2)-like intermediate species were generated at the middle temperature range and Ga–N bonds formed at high temperature along with dissociation of CO and CO2.

The rasoncogene products (H-Ras, K-Ras, and N-Ras) have been regarded as some of the most promising targets for anticancer drug discovery because their activating mutations are frequently found in human cancers. Nonetheless, molecular targeted therapy for them is currently unavailable. Here, we report the discovery of a small-molecule compound carrying a naphthalene ring, named KBFM123, which binds to the GTP-bound form of H-Ras. The solution structure of its complex with the guanosine 5′-(β,γ-imide) triphosphate-bound form of H-RasT35S (H-RasT35S·GppNHp) indicates that the naphthalene ring of KBFM123 interacts directly with a hydrophobic pocket located between switch I and switch II and allosterically inhibits the effector interaction by inducing conformational changes in switch I and its flanking region in strand β2, which are directly involved in recognition of the effector molecules, including c-Raf-1. In particular, Asp38 of H-Ras, a crucial residue for the interaction with c-Raf-1 via the formation of a salt bridge with Arg89 of the Ras-binding domain (RBD) of c-Raf-1, shows a drastic conformational change: its side chain orients toward the opposite direction. Consistent with these results, KBFM123 exhibits an activity to inhibit, albeit weakly, the association of H-RasG12V·GppNHp with the c-Raf-1 RBD. The binding of the naphthalene ring to the hydrophobic pocket of H-RasT35S·GppNHp is further supported by nuclear magnetic resonance analyses showing that two other naphthalene-containing compounds with distinct structures also exhibit similar binding properties with KBFM123. These results indicate that the naphthalene ring could become a promising scaffold for the development of Ras inhibitors.

Parkinson's disease (PD) can lead to swallowing dysfunction, resulting in aspiration pneumonia. Among the types of swallowing disorders, a characteristic and serious problem associated with PD is silent aspiration due to pharyngeal and laryngeal hypoesthesia.

Two-dimensional (2D) electrolytes have intrigued researchers due to their anisotropic structures and unconventional properties. This report introduces the bottom-up synthesis of a polymer-based 2D electrolyte utilizing a metal–organic framework (MOF), [Ni(Hbtc)(bpy)]n(1), with a pillared-layer structure as a template. Crosslinking polymerization of methyl methacrylate (MMA) within MOF nanochannels comparable in size to the vinyl monomer resulted in poly(methyl methacrylate) (PMMA) monosheets. Following hydrolysis of this precursor polymer successfully gave 0.6 nm thick poly(methacrylic acid) (PMAA) sheets. Despite the crosslinked structure, the obtained polymer (PMAA-1) is highly soluble as monosheets dispersed in water at a pH higher than 5.7. PMAA-1shows viscous liquid-like nature with the addition of the Al3+ion, which inherently promotes hydrogel formation of conventional linear PMAA. The topological effects of the 2D polyanion network structure on the uptake of cations in the matrix are likely responsible for these remarkable solution and viscoelastic properties of PMAA-1.

Hybrid endoscopic resection with an endo-knife and snare (HYB-ER) has been used to overcome the difficulties of endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD). This study clarified the role of HYB-ER in colorectal neoplasm treatment.

Recently, mesenchymal stem cells have demonstrated a potential for neurotrophy and neurodifferentiation. We have recently isolated mobilized dental pulp stem cells (MDPSCs) using granulocyte-colony stimulating factor (G-CSF) gradient, which has high neurotrophic/angiogenic potential. The aim of this study is to investigate the effects of MDPSC transplantation on peripheral nerve regeneration. Effects of MDPSC transplantation were examined in a rat sciatic nerve defect model and compared with autografts and control conduits containing collagen scaffold. Effects of conditioned medium of MDPSCs were also evaluated in vitro. Transplantation of MDPSCs in the defect demonstrated regeneration of myelinated fibers, whose axons were significantly higher in density compared with those in autografts and control conduits only. Enhanced revascularization was also observed in the MDPSC transplants. The MDPSCs did not directly differentiate into Schwann cell phenotype; localization of these cells near Schwann cells induced several neurotrophic factors. Immunofluorescence labeling demonstrated reduced apoptosis and increased proliferation in resident Schwann cells in the MDPSC transplant compared with control conduits. These trophic effects of MDPSCs on proliferation, migration, and antiapoptosis in Schwann cells were further elucidated in vitro. The results demonstrate that MDPSCs promote axon regeneration through trophic functions, acting on Schwann cells, and promoting angiogenesis.

ANDREW COYNE'S ARTICLE has my blood boiling ('The tipping plague,' Help, March 31). He wants us to believe that he's concerned about the seemingly out-of-control spread of tipping, but the [...]

Dental pulp stem cell (DPSC) subsets mobilized by granulocyte-colony-stimulating factor (G-CSF) are safe and efficacious for complete pulp regeneration. The supply of autologous pulp tissue, however, is very limited in the aged. Therefore, alternative sources of mesenchymal stem/progenitor cells (MSCs) are needed for the cell therapy. In this study, DPSCs, bone marrow (BM), and adipose tissue (AD)-derived stem cells of the same individual dog were isolated using G-CSF-induced mobilization (MDPSCs, MBMSCs, and MADSCs). The positive rates of CXCR4 and G-CSFR in MDPSCs were similar to MADSCs and were significantly higher than those in MBMSCs. Trophic effects of MDPSCs on angiogenesis, neurite extension, migration, and anti-apoptosis were higher than those of MBMSCs and MADSCs. Pulp-like loose connective tissues were regenerated in all three MSC transplantations. Significantly higher volume of regenerated pulp and higher density of vascularization and innervation were observed in response to MDPSCs compared to MBMSC and MADSC transplantation. Collagenous matrix containing dentin sialophosphoprotein (DSPP)-positive odontoblast-like cells was the highest in MBMSCs and significantly higher in MADSCs compared to MDPSCs. MBMSCs and MADSCs, therefore, have potential for pulp regeneration, although the volume of regenerated pulp tissue, angiogenesis, and reinnervation, were less. Thus, in conclusion, an alternative cell source for dental pulp/dentin regeneration are stem cells from BM and AD tissue.

The article looks at suggested summer activities for the family. Topics mentioned include tips for introducing little children to sleeping outdoors and camping, suggested outdoor summer activities including open-water swimming, orienteering and geocaching, and exploring a provincial park, and challenges encountered by parents while travelling with their children.

The collaborative learning remarks that were triggers of the past active discussion, which is called discussion promotion remark, can also be effective in the current discussion situation whose speech patterns are similar. The objective of this research is to construct the advising system for the collaborative learning which utilises discussion promotion remarks of the past collaborative learning as advice to solve current inappropriate situation. Discussion consists of various characteristics, so it is difficult to define appropriate speech pattern for each type of discussion promotion remark. This research introduces case-based reasoning approach to extract past discussion promotion remark which can solve current inappropriate situation. This paper describes two of the steps to accomplish the case-based reasoning system. First, several parameters that characterise the discussion situation are introduced and attached to past discussion promotion remarks. Second, discussion promotion remark database is constructed as a decision tree based on the attached parameters.

We demonstrate that NH3nitridation of sapphire substrates effectively suppresses cracks in AlN epilayers. The sapphire nitridation promoted three-dimensional (3D) growth at the initial stage, in contrast to the 2D growth mode on non-nitrided sapphire. The coalescence of 3D columnar grains in the process created voids, which act as strain absorber and thus crack-free thick epilayers were obtained. The control of nitridation period is also found important. The optimum nitridation period realized an atomically-smooth epilayer with superior structural quality. On the other hand, the least nitrided sapphire exhibited high twist mosaic of the grains which complicate the epilayer smoothing process, and the longer nitridation promoted slow recovery of a smooth epilayer.

Intelligent Tutoring System (ITS) is a learning support system which provides an individual learning environment to students. ITS gives learning contents to students according to the learning strategy based on their understanding degree. Many traditional ITSs, however, have static predefined contents without regard to individual preferences. It causes that students lose their motivation. Our research objective is to develop a system which automatically generates multiple–choice cloze exercises from input text according to the student's understanding. Our target is supporting individual study of learning English grammar. In this paper, we propose a representation method of English grammar by Part–of–Speech (POS) tags and specific words as units and the semantic relations of the units. In addition, the calculation method for estimating understanding degree of the student and the learning strategy for generating the next exercise proposed regarding the understanding degree of student and the relations of grammar rules.

When Chinese students study Japanese, they sometimes find it difficult to understand the grammar of Japanese correctly because Chinese and Japanese use the same Chinese characters in a different way. Our research focuses on the students’ exercises of translating Chinese sentences into Japanese, and enables students to acquire correct knowledge based on translated sentences. For this objective, our Chinese-Japanese translation process is divided into three steps: translation of words, ordering of words, and addition of functional expressions to complete the sentence. The system compares translated sentences written by students with the correct answer preset in the system and specifies the step in which students might be mistaken. Then, the system gives a result of evaluating the translated sentence with the explanation of incorrect translation. Since the usage of Japanese functional expressions is the most difficult for the Chinese students translating a sentence, our system supports students learning Japanese functional expressions by considering the factors of the mistaken functional expressions. According to the mistake information, the system selects another question from the question database, which has the same factor as the mistaken functional expression. Experimental results showed that our system was effective in detecting the mistakes, especially mistakes of words and Japanese functional expressions.

The article offers suggestions to women on how to celebrate Christmas holidays in good spirits and stress-free. It suggests to personalize holidays and spend days with friends who bring happiness. It states that if one does not want to spend too much time with the family, one should set a limit on the family time. Life coach Ellen Smith suggests one to take a team approach while hosting an event and let the guests bring side dishes and desserts, while main dish can be prepared at one's place.

The article presents the views of several Canadian women on turning 30 years old. It is stated that for a generation of women who grew up viewing equality as their right, today's 30s are building on the feminist legacy created by the past generations. Producer Fiona Solon says when she turned 30, she realized she had the potential to do anything.